Synuclein aggregation in dopaminergic neurons is a hallmark of Parkinson's disease (PD) pathology. The most common genetic risk factor for PD is glucocerebrosidase (GBA) mutation with as many as 7% of PD patients carrying this mutation. Work to date has primarily focused on the loss of GBA enzymatic activity that could contribute to ?-synuclein accumulation and/or aggregation. However, evidence has indicated that improperly folded mutant GBA could also contribute to the ?-synuclein aggregation independent of the loss of enzymatic activity. Our central hypothesis is that mutant GBA is mistargeted from lysosomes to the cytosol, which interferes with ?-synuclein degradation by chaperone-mediated autophagy (CMA). We will test this hypothesis with a combination of biochemical, cell biological, mouse genetics, and neuropath logical techniques in several systems including isolated lysosomes, mouse models, post-mortem human brain, and fibroblasts from patients' skin biopsy. Our research strategies are divided into three specific aims: Aim 1 will investigate the mechanism by which mutant GBA attenuates CMA and leads to ?-synuclein accumulation by examining each step of CMA in an in vitro system using isolated lysosomes, purified mutant GBA and ?- synuclein proteins. Aim 2 will study whether CMA alteration occurs in GBA-mutant mouse models by using a novel CMA reporter in neuronal cultures and determine whether the protein levels of CMA machinery components change in GBA-mutant mouse models and post-mortem PD brain with GBA mutations. Aim 3 will determine CMA activity using the reporter in the skin fibroblasts from PD patients with GBA mutations, PD patients without GBA mutations, and age-matched controls. These data will provide evidence on whether mutant GBA causes CMA dysfunction, leading to ?-synuclein aggregation, and whether CMA dysfunction is a typical feature of PD. The proposed study could contribute to the future identification of mechanism-based biomarker and therapeutic targets. This K08 proposal also outlines a detailed 5-year training program with specific formal coursework and structured mentoring for the candidate, Sheng-Han Kuo, M.D. The proposed work will be carried out in the Department of Neurology at Columbia University, an excellent environment for training physician-scientists. He will receive the necessary training under the mentorship of Drs. David Sulzer, Ana Maria Cuervo, and Karen Marder, world-renowned investigators in the PD field and acquire necessary skill set to become an independent researcher. The long-term goal of the candidate is to be a translational physician-scientist to investigate the mechanism underlying PD pathology and to develop clinical applicable biomarkers for PD.